An information explosion on the number of functional RNA
molecules expressed in cells and the mechanisms by which they control gene
expression1,2 is driving the formation and funding of biotechs
developing oligonucleotide-based therapeutics. Pharmas and big biotechs are
carving up the space via partnerships that add multiple nucleic acid-based
mechanisms and technologies to their drug discovery toolboxes.

The
newest additions to the nucleic acid platform include noncoding RNA targets
such as microRNAs and long noncoding RNAs (lncRNAs). Both types of molecule
create the opportunity to selectively turn on expression of a given gene,
potentially achieving biological and therapeutic outcomes that no other drug platform
can.

Small
molecules that interfere with the activity of chromatin-modifying enzymes can
lead to the activation of many genes, but no small molecule or biologic has
been shown to recapitulate the single-gene specificity achievable via the
complementary base pairing of oligonucleotide-based modalities.

Although
miRNAs and lncRNAs offer new targets and mechanisms, the modalities that target
these molecules share one important feature with prior oligonucleotide-based
therapeutics-the reliance on interactions between complementary nucleotides.
Thus, therapeutics aimed at these new molecules share many of the challenges
encountered with other nucleic acid-based agents.

These
include delivering the drug to many tissue types, achieving levels of target
engagement necessary for therapeutic effects and selecting and validating
targets best suited to the modality.

In the
beginning

Attempts to use antisense technology as a
therapeutic began in the late 1980s. The rationale was clear-find a gene that
is causing problems, synthesize a strand of nucleic acid that will bind to the
mRNA and use that molecule to shut down the gene. However, a host of practical
issues got in the way.

Oligonucleotide-based
molecules are not drug like; thus, they require chemical modifications that
improve their pharmacokinetics (see"Drug-like
chemical modifications to nucleic acids"). In addition, there are multiple
delivery challenges. For example, cells need to be coaxed into taking up
oligonucleotide-based therapeutics and releasing enough of them from
intracellular vesicles to elicit a therapeutic effect without activating an
immune response.

Finally,
the mechanism of action of some of these oligonucleotide molecules involves
enzymatic degradation of the oligonucleotide-target complex. For example,
antisense molecules elicit RNase H-mediated degradation of their targets.3
Such mechanisms limit the range of pharmacological improvements that can be
made to the molecules before they become unrecognizable to the relevant
enzymes.

"Isis
invented the antisense platform as a viable drug discovery and development
approach," said Brett Monia, the company's SVP of antisense drug
discovery.

"In
the early years, we needed to partner early so that we could build a new
technology. However, having a partner develop your drugs, especially something
as different as antisense, is much less efficient and less profitable. Today,
we hold on to our drugs much longer. The flexibility to partner a program at
the optimal time is a huge advantage," Monia added.

Isis
now has a pipeline of 19 antisense molecules in the clinic and 1-mipomersen-on the market.

This
year, the FDA approved mipomersen, which targets apolipoprotein B-100 (APOB-100) mRNA, as an
adjunct to lipid-lowering medications and diet to treat patients with
homozygous familial hypercholesterolemia.

"The
approval of mipomersen was a great moment for the advancement of
oligonucleotide therapeutics," said John Maraganore, CEO of Alnylam Pharmaceuticals Inc.

Isis
continues to be a prolific deal maker. Ten of its molecules in clinical testing
are partnered, and the biotech recently announced a blockbuster neurology
alliance with Biogen Idec Inc.

Santaris'
locked nucleic acid (LNA) technology was one of the first and remains one of
the best ways to improve the drug-like properties of oligonucleotides. LNAs
increase an oligonucleotide's thermal stability, selectivity for the
complementary target sequence and resistance to digestion by nucleases.

At least seven companies developing
oligonucleotide-based drugs have licensed the technology from or partnered with
Santaris, which has an internal pipeline of six antisense molecules in the
clinic.

Rather than knocking down gene expression, at least two
companies are taking a distinct approach-using antisense oligonucleotides to
induce exon skipping. In this approach, an antisense oligonucleotide blocks
access of the splicing machinery to the target, which is unspliced pre-mRNA,
resulting in the adjacent exon being skipped or omitted from the mature mRNA.

Omission of an exon that does not disrupt the
translational reading frame results in the production of a smaller but at least
partially functional protein.

For
example, in most cases of Duchenne muscular dystrophy (DMD), a deletion in the dystrophin gene disrupts the
translational reading frame, thus inhibiting expression of the protein.
Antisense oligonucleotides that induce skipping of exon 51 partially rescue
dystrophin function. However, it is not yet clear if this approach is working
in DMD.

In
September, GlaxoSmithKline plc and Prosensa Holding N.V. announced that the
biotech's drisapersen, an RNA-based
antisense drug that induces exon 51 skipping in the dystrophin gene,
failed in a Phase III study.

Moderna Therapeutics Inc., founded in 2010 by Flagship Ventures, is developing oligonucleotide-based
therapeutics that directly encode the expression of human proteins in vivo. The
biotech is not using or targeting noncoding RNAs and instead has developed a
chemically unique RNA-based platform.

In 2001, small interfering RNAs, which feed
into the RNAi pathway (see"The RNAi pathway"), were shown to lead to mRNA
degradation in cultured human cells.4 In contrast with their single-stranded
antisense counterparts, siRNA therapeutics are double stranded.

Companies
developing siRNA-based therapeutics have been particularly challenged by
delivery, as double-stranded oligonucleotides are notably worse than their
single-stranded counterparts at accessing the intracellular space in which most
RNA targets are found. Indeed single-stranded antisense modalities can often be
delivered in saline, whereas double-stranded molecules require a vehicle to get
them across the plasma membrane.

Alnylam
has pioneered the development of siRNA therapeutics, with a focus on orphan
indications in which drugs need to be targeted to the liver, which is where
lipid nanoparticles carrying siRNA can accumulate.5

According
to Maraganore, "Many orphan diseases are caused by mutations in genes that
cannot be targeted by other therapeutic modalities such as small molecules and
monoclonal antibodies."

In
March, Alnylam started Phase I testing of ALN-TTRsc, an siRNA that
targets transthyretin (TTR) mRNA to treat
TTR-mediated amyloidosis (ATTR).

"We've
shown that we can knock down TTR by up to 94% in human studies. The drug
was found to have a very encouraging safety profile. In short, GalNAc-siRNA
opens up the door for RNAi therapeutics in a much broader range of clinical
indications," said Maraganore.

The space that has seen the most new company
formation in recent years is miRNAs, which are naturally occurring noncoding
RNAs that function like siRNAs in the RNAi pathway. They are about 22
nucleotides in length and regulate the expression of gene networks by
interacting with the 3ʹ untranslated region of mRNAs and blocking
translation or regulating degradation of their targets. Dysregulation of miRNAs
was first linked to human disease in 2002.6

Anti-miRNAs
are synthetic antisense oligonucleotides that target miRNAs and thereby inhibit
their function, usually resulting in the upregulation of gene expression.
Similar to antisense molecules targeting mRNAs, anti-miRNAs are often single
stranded and with the right nucleic acid chemistry can be delivered in saline.

miRNA
mimics are synthetic, double-stranded oligonucleotides designed to replace
miRNA function. Similar to siRNAs, these molecules inhibit gene expression.
Because they are double stranded, delivery vehicles are generally necessary for
these molecules to reach intracellular targets.

In
2007, Isis and Alnylam combined their miRNA-directed programs and formed Regulus Therapeutics Inc., the first purely
miRNA-based therapeutics company.7 Regulus received exclusive rights
to both Isis' and Alnylam's technologies covering the therapeutic application
of anti-miRNAs.

Last
year, Regulus raised $50.9 million in an IPO, $25 million from the concurrent
sale of stock to AstraZeneca plc and Isis and $5 million from the sale
of a convertible note to Biogen.

In
2014, Regulus expects to submit an IND for lead compound RG-101, a GalNAc-conjugated anti-miRNA
targeting hepatocyte miR-122, to treat HCV.

Mirna
is focused on miRNA replacement therapies for cancer. The company's lead
candidate is MRX34, a liposomally formulated miR-34 mimic that is in
Phase I testing to treat liver cancer.

miRagen
is using Santaris' LNA technology to develop miRNA-targeted therapies. miRagen
has a deal with Servier in cardiovascular disease, and its programs
are in preclinical development.

MiReven Pty. Ltd. is a newer entrant in the miRNA
space. The company was formed in 2010 and has miR-7 mimics in preclinical development to
treat cancer. It partnered with Silence Therapeutics plc last year for the
latter's lipid-based delivery technology.

Santaris
was the first company to bring a miRNA-targeting molecule into the clinic. The
company's miravirsen, an LNA-modified
anti-miRNA that targets hepatocyte miR-122, is in Phase II testing to treat
HCV. In 2013, Santaris published Phase IIa data showing that miravirsen
resulted in a dose-dependent reduction in HCV RNA levels.8

Santaris
also has anti-miRNAs in preclinical development.

The long
noncoding road

Even newer than miRNAs are lncRNAs, which
are noncoding RNA targets that can positively and negatively regulate gene
expression.9

Studies
of newly discovered lncRNAs are revealing previously unknown regulatory
mechanisms that control gene expression, although strategies for targeting or
mimicking these molecules share many of the chemical, biological and logistical
traits of siRNA and antisense therapeutics.

Two
studies formed the foundation for the first companies focused on therapeutics
targeting lncRNAs.

In
2010, scientists in the laboratory of Jeannie Lee showed that a histone
methyltransferase called polycomb repressive complex 2 (PRC2) interacted with more than 9,000
RNAs, many of which had not previously been annotated.10 Based on the
findings, the group proposed that lncRNAs can drive target-specific
interactions between chromatin-modifying complexes and genomic DNA.

In
2011, Lee cofounded RaNA Therapeutics Inc., which received
seed funding from Atlas Venture. The biotech is developing oligonucleotide-based
therapeutics to disrupt lncRNAs that recruit PRC2 to promoters. The compounds
control the expression of therapeutically relevant proteins by specifically
upregulating their expression.

"Our
focus is on the selective upregulation of a therapeutic protein that has
already been validated as important in disease," said Art Krieg,
president, CEO and cofounder of RaNA.

RaNA
has in vitro proof of concept that an oligonucleotide-based agent can
specifically alter expression of a target gene by disrupting interactions
between lncRNA and PRC2.

The
company has programs in spinal muscular atrophy and Friedreich's ataxia and
hopes to advance a molecule into the clinic in 2015, said Krieg.

In
January 2012, RaNA raised $20.7 million in a series A round led by Atlas, SR One and Monsanto Co. RaNA has a proprietary database of more
than 50,000 PRC2-associated lncRNAs and owns 28 patent applications.

The
second seminal study in the lncRNA field was published in 2012 by a group led
by Claes Wahlestedt. The team showed that antagonizing lncRNAs that function as
natural antisense transcripts (NATs) de-repressed and thus activated the
expression of a specific gene.11 The work also showed that a nucleic
acid-based modality called an antagoNAT could achieve interference with NATs in
vivo.

In
2008, Wahlestedt, then a professor of molecular therapeutics and adjunct
professor of molecular and integrative neuroscience at Scripps Florida, cofounded Curna Inc. with a
$350,000 loan from the town of Jupiter, Fla., to commercialize the antagoNAT
strategy for upregulating expression of therapeutically relevant proteins.

In
February 2011, Opko Health Inc. acquired Curna for $10 million.
About 2 years later, RXi Pharmaceuticals Corp. acquired Opko Health's RNAi-related assets for about $7
million in stock and up to $50 million in development and commercialization milestones
for each product. At the time it was acquired by Opko Health, Curna had filed
for at least 90 patents.

Big pharma's
big place

Over the now several
decades-long history of nucleic acid therapeutics, big pharma has had an
on-again, off-again relationship with the technology.

According
to Maraganore, "In the 2005-2009 period, big pharma made big bets on RNAi
technology platforms. Unfortunately, some of the big pharma technology efforts
have not been so successful, at least as measured by IND filings, of which
there are none."

In
2010, Roche abandoned its internal efforts to develop
RNAi as a drug platform. In early 2011, Pfizer Inc. announced that it was cutting its RNAi drug
discovery programs among others as part of a restructuring plan. Abbott Laboratories soon followed suit.

Also
in 2011, Merck & Co. Inc. shut down the San
Francisco RNAi research site that had been Sirna
Therapeutics Inc., which it had acquired for $1.1 billion in
2006, but said it remained committed to RNAi technology. Merck and Novartis AG are the only big pharmas that have
disclosed internal, RNA-directed therapeutics programs.

In
October of this year, Merck said it was restructuring R&D. The pharma did
not say whether cuts will include its RNAi platform.

According
to Krieg, "Pharma is becoming more modality agnostic instead of just
summarily dismissing oligos as drugs. They are primarily now just looking at
these agents as drugs."

Maraganore
agreed. "RNAi therapeutics partnerships will be focused on product
alliances. Pharma has recently grown their interest in the RNA therapeutics
space but with an interest in products, not technology," he said.

Access this BioCentury Innovations article Cover Story for your individual use via a permanent link that allows you to read or print the article, and any sub-articles, charts, tables and/or graphs related to this specific story: $50.
The article link will be posted on the purchase transaction web page, and also emailed to you with your purchase confirmation.

Purchase This Article for Limited One-Time Distribution and Posting to Your Website :

Receive a formatted PDF reprint of this article, including any sub-articles, charts, tables and/or graphs related to this specific article, with rights for limited one-time redistribution and posting to your website: $750. Please allow 24-48 hours for delivery.

Purchase Options

Purchase this article for individual use $50 USDPurchase this article for limited one-time distribution and website posting $750 USD